Pharmacologic Inhibition of the Menin MLL Interaction ... · Chromatin immunoprecipitation A total of 1.5 106 HepG2 cells plated in 10-cm dish were treated with DMSO (0.25%) or MI-503

Small Molecule Therapeutics

Pharmacologic Inhibition of the Menin–MLLInteraction Leads to Transcriptional Repressionof PEG10 and Blocks Hepatocellular CarcinomaKatarzyna Kempinska, Bhavna Malik, Dmitry Borkin, Szymon Klossowski,Shirish Shukla, Hongzhi Miao, Jingya Wang, Tomasz Cierpicki, and Jolanta Grembecka

Abstract

Hepatocellular carcinoma (HCC) accounts for approximately85%ofmalignant liver tumors and results in 600,000 deaths eachyear, emphasizing the need for new therapies. Upregulation ofmenin was reported in HCC patients and high levels of menincorrelate with poor patient prognosis. The protein–protein inter-action between menin and histone methyltransferase mixedlineage leukemia 1 (MLL1) plays an important role in the devel-opment of HCC, implying that pharmacologic inhibition of thisinteraction could lead to new therapeutic strategy for the HCCpatients. Here, we demonstrate that themenin–MLL inhibitorMI-503 shows antitumor activity in in vitro and in vivomodels of HCCand reveals the potential mechanism of menin contribution toHCC. Treatment with MI-503 selectively kills various HCC celllines and this effect is significantly enhanced by a combination of

MI-503 with sorafenib, the standard-of-care therapy for HCC.Furthermore, MI-503 reduces sphere formation and cell migra-tion in in vitroHCCmodels. When applied in vivo, MI-503 gives astrong antitumor effect both as a single agent and in combinationwith sorafenib inmice xenograftmodels ofHCC.Mechanistically,treatment withMI-503 downregulates expression of several genesknown to play a critical role in proliferation and migration ofHCC cells, including PEG10, and displaces the menin–MLL1complex from the PEG10 promoter, resulting in reduced H3K4methylation and transcriptional repression. Overall, our studiesreveal a mechanistic link between menin and genes involved inHCC and demonstrate that pharmacologic inhibition of themenin–MLL interaction might represent a promising therapeuticapproach for HCC. Mol Cancer Ther; 17(1); 26–38. �2017 AACR.

IntroductionHepatocellular carcinoma (HCC) accounts for approximate-

ly 85% of primary malignant liver tumors and is the fifth mostcommon cancer in the world, leading to over 600,000 deathseach year (1, 2). The majority of HCC patients are diagnosed ata late stage of the disease, when curative therapies are no longereffective (3), and most patients die about 6 months afterdiagnosis (1, 4, 5). Furthermore, patients who initially respondto curative therapies frequently experience disease recurrence,resulting in an overall 5-year survival of only 7% (6), whilepatients with surgically resectable tumors have survival ratesstill below 40% (7). All these findings strongly support the needfor new and more effective therapies for HCC. One recent newtreatment strategy for advanced HCC patients is the use of themultitargeted kinase inhibitor sorafenib (3), which blocks the

activity of Raf serine/threonine kinases and various receptortyrosine kinases (8). Sorafenib inhibits the proliferation ofHCC cells and blocks tumor angiogenesis, resulting in anincrease of about three months in the median survival of HCCpatients (8). However, sorafenib has only limited clinicalefficacy with undesired side effects (9), strongly implying theneed for more efficacious therapies for HCC.

Recently it has been demonstrated that menin, a scaffoldprotein encoded by the multiple endocrine neoplasia type 1(MEN1) gene, is involved in promoting hepatocellular carcinoma(10). Menin is a highly specific binding partner of mixed lineageleukemia 1 (MLL1, also known as KMT2A; refs. 11, 12), a histonemethyltransferase that catalyzes the trimethylation of H3K4(H3K4me3) (13), and is required for the recruitment of theMLL1complex to the target genes (14). Menin upregulation wasreported in HCC patients, and high levels of menin correlatewith poor patient prognosis (10). Indeed, menin knockdownsubstantially represses HCC cell proliferation, blocks colonyformation, and inhibits tumor growth in the xenograft model ofHCC (10). Likewise, heterozygous loss of Men1 reduces devel-opment of HCC in vivo (10).

The function of menin in liver tumorigenesis has been linkedto the transcriptional regulation of the Yes-associated protein(Yap1), an important oncogene in HCC (15) and a down-stream target of the Hippo pathway (16, 17). Menin occupancyat the promoter region of Yap1 has been noted to coincide withH3K4me3, a histone mark regulated by MLL1 (10), implying apotential involvement of the menin–MLL1 interaction in HCCdevelopment. We, and others, have previously shown a criticalrole of the menin interaction with MLL1 and/or MLL fusion

Department of Pathology, University of Michigan, Ann Arbor, Michigan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

K. Kempinska and B. Malik contributed equally to this article.

Current address for J. Wang: Medimmune, LLS, Gaithersburg, Maryland.

Corresponding Authors: Jolanta Grembecka, Department of Pathology, Uni-versity of Michigan, 1150 West Medical Center Dr, MSRB I, Room 4510D, AnnArbor, MI 48108. Phone: 734-615-9319; Fax: 734-763-4851; E-mail:[email protected]; and Tomasz Cierpicki, [email protected]

doi: 10.1158/1535-7163.MCT-17-0580

�2017 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 17(1) January 201826

on December 26, 2020. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst November 15, 2017; DOI: 10.1158/1535-7163.MCT-17-0580

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-17-0580&domain=pdf&date_stamp=2017-12-13

http://mct.aacrjournals.org/

proteins in acute leukemias with translocations of theMLL gene(12, 18), as well as in solid tumors, including metastaticprostate cancer (19). All these studies imply that the menin–MLL1 interaction might play a more general role in cancer,including liver tumorigenesis; therefore inhibition of this inter-action with small molecules might represent a novel therapeu-tic approach for HCC treatment. On the other hand very limitedeffect observed in HCC cells following treatment with a weakmenin–MLL inhibitor MI-1 (no significant effect on cell via-bility in liquid culture and <25% inhibition of colony forma-tion and cell migration when applied as a single agent at 4mmol/L; ref. 20) raises questions regarding the potential ther-apeutic value of blocking the menin–MLL1 interaction as atreatment for liver cancer.

We have recently reported very potent small-molecule inhibi-tors of the menin–MLL1 interaction with optimized drug-likeproperties, includingMI-503 (IC50¼ 14 nmol/L, Kd¼ 9 nmol/L),which demonstrated very potent activity in both in vitro and in vivomodels of MLL leukemia (18, 21–23) as well as in castration-resistant prostate cancer (19). As the in vitro potency of MI-503 is>140-fold better than MI-1 and it demonstrates strong andselective activity in cancer cells, we selected MI-503 to study theeffect of pharmacologic inhibition of the menin–MLL interactionin liver cancer. Here, we performed a systematic evaluation of theMI-503 in HCC models to assess whether pharmacologic inhi-bition of the menin–MLL1 interaction might represent a newtherapeutic strategy for liver cancer. Treatment withMI-503 had avery pronounced effect in various models of HCC, both in vitroand in vivo, and this effect was enhanced when MI-503 wascombined with sorafenib, the current standard-of-care therapyin patients with advanced HCC. Pharmacologic inhibition ofmenin-MLL1 by MI-503 strongly downregulated the expressionlevel of genes previously linked to HCC, including PEG10 (24,25), thereby providing a new mechanistic insight into the role ofmenin–MLL1 and H3K4me3 in HCC. Overall, our findings dem-onstrate that pharmacologic inhibition of themenin–MLL1 inter-action canblockprogressionofHCC, thus validating this protein–protein interaction as an attractive target for therapeutic interven-tion in liver cancer.

Materials and MethodsChemistry

Chemical synthesis and chemical characterization of MI-503and MI-372 compounds have been described previously (18).

Cell cultureThe HepG2 (low metastatic; ref. 26) and Hep3B (low meta-

static; ref. 27) humanHCC cell lines were obtained from ATCC in2014 while the remaining cell lines (MHCC97, PLC/PRF/5,SNU449, and SNU423) were received from Dr. Ilona Kryczek(University of Michigan, Ann Arbor, MI). HepG2, Hep3B, andPLC/PRF/5 cells were maintained in Eagle's minimum essentialmedium (EMEM; ATCC) with 10% FBS and 1% penicillin/strep-tomycin (Gibco) antibiotics. SNU449, SNU423, and MHCC97were cultured in RPMI1640 media with 10% FBS and 1% pen-icillin/streptomycin (Gibco). ASC52 cell line was received fromDr. Elizabeth Lawlor (University ofMichigan, AnnArbor,MI) andcultured in mesenchymal stem cell (MSC) basal medium withMesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derivedMSC Low Serum Components and G418 (ATCC). All celllines were used in the described experiments before reaching the

10th passage after thawing the cells out and were negative formycoplasma prior performing these studies as assessed by theTOKU-E PCR Mycoplasma Detection Kit.

Cell viability assayCell viability assays are described in the Supplementary

Information.

Cellular thermal shift assayCellular thermal shift assay (CETSA) was performed as

described previously (19, 28). Experimental details are providedin the Supplementary Information.

Real-time qPCRCells were plated at a concentration of 0.25 � 106 cells/mL in

the 12-well plate in duplicates and treated with MI-503 or DMSOfor 6 and/or 13 days followed by RNA isolation according to themanufacturer's protocol (RNeasy kit, Qiagen), RNA reverse tran-scription to cDNA, andqPCR (see Supplementary Information fordetails).

RNA Sequencing studiesHepG2 cells were plated in the 12-well plates at the initial

concentration of 0.4 � 106 cells/mL and treated with 3 mmol/LMI-503 or DMSO (0.25%) in triplicates. After 3 days of treat-ment, cells were trypsinized, counted, and viable cell numberwas adjusted to the original concentration. Media were changedand compound or DMSO was resupplied at that time. Cellswere harvested after 3 more days of incubation. Total RNA wasisolated from cells using RNeasy kit (Qiagen), amplified, andquality was assessed using the TapeStation (Agilent). Sequenc-ing was performed at the University of Michigan DNA Sequenc-ing Core. Samples with RIN (RNA Integrity Numbers) of 8 orgreater were prepped using the Illumina TruSeq mRNA kit(Illumina). RNA was converted to mRNA using a polyA puri-fication. cDNA library was created using reverse transcriptase,barcoded, and sequenced using 4 samples per lane on a HiSeq2000 (Illumina) in High Output mode. Sequenced reads werealigned to human reference genome using Bowtie and Tophat(version 2.0.3). Differential gene expression analysis was doneusing program Cuffdiff. Genes with P < 0.05 and a fold changegreater that 2-fold were considered significant. Gene Set Enrich-ment Analysis (http://www.broadinstitute.org/gsea/index.jsp)was performed on gene expression of MI-503–treated versusDMSO-treated cells.

Sphere formation and cell migration assaysSphere formation and cell migration assays are described in

Supplementary Information.

In vitro combination studiesHepG2 and Hep3B cells were plated at a concentration of

0.45 � 105 cells/mL in a 24-well plate and treated with 0.25%DMSO, sorafenib, MI-503, or both compounds. After 4 days (forHepG2) or 5 days (forHep3B) of incubation cells were transferredto the 96-well plate in quadruplicates. The MTT cell proliferationassay kit (Roche) was then applied. Plates were read for absor-bance at 570 nmusing a PHERAstar BMGmicroplate reader. Druginteractions were analyzed by CalcuSyn program (Biosoft) basedon the analytical method of Chou and Talalay (29).

Menin–MLL Inhibition in Hepatocellular Carcinoma

www.aacrjournals.org Mol Cancer Ther; 17(1) January 2018 27



http://www.broadinstitute.org/gsea/index.jsp


Chromatin immunoprecipitationA total of 1.5 � 106 HepG2 cells plated in 10-cm dish were

treated with DMSO (0.25%) or MI-503 at 4 mmol/L, 2 mmol/L,and 1 mmol/L concentrations for 6 days. After 3 days of incuba-tion, cell number was adjusted to the starting concentration,media were changed, and fresh compound was added. Following6 days of incubation, cells were fixed with 1% paraformaldehydefor 10 minutes at room temperature. Cells were then quenchedwith glycine for 5 minutes at room temperature. ChIP wasperformed using Magna ChIP A/G kit (Millipore) according tothe manufacturer's protocol. The cell lysate was prepared andequal amounts of DNA isolated from either DMSO or MI-503–treated samples was immunoprecipitated using antibodiesagainst Menin (Bethyl Laboratories), MLL1 (Millipore), H3(Abcam), H3K4me3 (Abcam), and IgG (Millipore) overnight at4�C. TheDNAwas eluted and subjected to quantitative PCR usingSYBR Green primers for PEG10 (see Supplementary Informationfor primer sequences and corresponding genomic referencecoordinates).

Animal studiesAll animal studies were performed in compliance with the

guidelines of the University of Michigan Committee for Use andCare of Animals and Unit for Laboratory Animal Medicine(ULAM) under the protocol PRO00005043. Freshly thawedHepG2 or Hep3B cells (ATCC) were cultured in EMEM with10% FBS and 1% penicillin/streptomycin antibiotics. On the dayof injection, cells were resuspended in serum-free medium at 1�107 cells/mL and mixed 1:1 with Matrigel. A total of 0.5 � 107

cells/mL were injected to the flank of the 6-week-old femaleathymic nude mice. When tumor size reached approximately100 mm3, mice were randomized into four groups with eachgroup containing 8–9 mice to provide over 80% power to detect50%effect.Micewere then treatedwith vehicle (25%DMSO, 25%PEG400, 50% PBS), MI-503 (dissolved in vehicle), sorafenib(dissolved in 12.5%Cremophor, 12.5%Ethanol and 75%water),or both compounds once daily at the designated doses usingintraperitoneal (vehicle and MI-503) or oral, (sorafenib) admin-istration. Bodyweight and tumor sizesweremonitored3 timesperweek. The tumor volumewas calculated according to the formula:tumor volume ¼ a � b2/2 (a, long diameter; b, short diameter).Treatment continued until the mean tumor size in the vehiclegroup reached approximately 1,200mm3. The experimenterswerenot blind to group assignment and outcome assessment.

Statistical analysisStudent t test (unpaired, two-tailed) was used to calculate

significance level between treatment groups to calculate P values.P values of less than 0.05 were considered significant. Graphgeneration and statistical analysis were performed using Graph-Pad Prism version 6.02 software (GraphPad).

Accession numbersRNA-seq data were deposited at the NCBI Gene Expression

Omnibus under the accession number GSE103327.

ResultsMenin is overexpressed in liver cancer cells

To understand the tumorigenic function of menin in hepato-cellular carcinoma, we compared the expression level of MEN1,

which encodesmenin, in normal liver and liver tumor samples byanalyzing the Oncomine datasets (30). Comparison of theMEN1expression level between the two sets of normal liver and HCCpatient samples (31) revealed a significant upregulation ofMEN1in the liver tumor samples (Fig. 1A, consistent with the previousstudies; ref. 10). We further investigated the role of menin in livercancer by assessing the level of menin in several human HCC celllines: HepG2, Hep3B, SNU449, SNU423, MHCC97, PLC/PRF/5.Western blot analysis confirmed that menin is expressed anddetectable in each of these HCC cell lines, although some vari-ability in the menin expression was observed (SupplementaryFig. S1A).

Studies by Xu and colleagues (10) support the importance ofthe menin interaction with MLL in hepatocellular carcinoma.Therefore, we used the panel of HCC cell lines to evaluate theexpression level of twoMLL familymembers,MLL1 (KMT2A) andMLL2 (KMT2B), which are both capable of strong interactionwithmenin (11). Interestingly, we foundmuch higher expression levelof MLL1 than MLL2 in all HCC cell lines tested (SupplementaryFig. S1B) which suggests that MLL1, rather than MLL2, plays animportant role in liver cancer through a direct interaction withmenin.

Menin–MLL inhibitor blocks proliferation of HCC cellsKnock down ofmenin substantially represses proliferation and

reduces colony formation in HCC cells (10). To determinewhether pharmacologic inhibition of the menin–MLL1 interac-tion affects growth of liver cancer cells, we used our recentlyreported and very potent menin–MLL1 inhibitor MI-503 (Sup-plementary Fig. S1C), which has demonstrated strong activity inMLL leukemia (18) and prostate cancer (19) models. Treatmentwith MI-503 inhibited the proliferation of HepG2 liver cancercells in a dose- and time-dependent manner, with a more pro-nounced effect observed upon prolonged treatment (over 7days; Fig. 1B). The need for a prolonged treatment in the HCCcells is consistent with the effects we observed in MLL leukemia(18) and prostate cancer cells (19). Further examination of MI-503 in a panel of HCC cell lines revealed a strongly decreasedviability of these cells after 7 and 12 days of treatment, with a half-maximal growth inhibitory (GI50) concentration ranging from0.5to 3.2 mmol/L after 12 days of treatment (Fig. 1C; SupplementaryTable S1). Importantly, treatment with a structurally similar butmuchweakermenin–MLL inhibitor, MI-372 (Supplementary Fig.S1C), did not show an effect on HCC cell proliferation (Supple-mentary Fig. S1D; Supplementary Table S1). These results validatethat targeted inhibition of the menin–MLL1 by MI-503 inhibitsthe growth of HCC cells. We have also tested the effects of MI-503treatment in normal adipose-derived ASC52 mesenchymal stemcells, which served as a negative control cell line, and observed nosubstantial effect on cell growth up to 6 mmol/L of MI-503 (Fig.1D; Supplementary Table S1). These data are consistentwithnoorlimited effect of MI-503 in other negative control cell lines as wereported previously (18, 19) and demonstrate specificity of MI-503 on proliferation of liver cancer or other menin–MLL–depen-dent cells (18). To assess whether cell growth inhibition correlateswithmenin engagement byMI-503,weperformed theCETSA.MI-503 treatment of HepG2 cells at 45�C induced a thermal stabi-lization of menin when compared with the DMSO-treated cells(Fig. 1E; Supplementary Fig. S1E), confirming that MI-503 bindsto menin in HCC cells.

Kempinska et al.

Mol Cancer Ther; 17(1) January 2018 Molecular Cancer Therapeutics28




MI-503 blocks cell migration and sphere formationin HCC cells

The menin–MLL1 interaction was shown to contribute to theaggressive nature of HCC (10). As cell migration is a property of

cancer cells associated with cancer metastasis, we assess the effectof menin inhibition on HCC cell migration using the cell culturewound closure assay (32) bymeasuring the rate of wound closureafter treatment with MI-503. The migration of both HepG2 and

Figure 1.

Effect of MI-503 menin–MLL inhibitor on HCC cell lines growth. A, MEN1 expression in normal versus tumor liver cells based on the analysis of the Oncominedatasets (30). B, Growth curves demonstrating the effect of MI-503 on cell growth in HepG2 cells. Viable cells were counted at days 3, 6, 9, and 13. Data arenormalized to DMSO-treated cells and represent mean of duplicates � SD. C, MTT cell viability assay performed in HCC cell lines after 12 days of treatmentwith MI-503. Data are normalized to the DMSO-treated cells and represent mean of quadruplicates � SD. D, MTT cell viability assay performed for MI-503 innormal adipose-derived mesenchymal stem cells, ASC52. Data are normalized to the DMSO-treated cells and represent mean of quadruplicates � SD. E, Resultsfrom the CETSA performed in HepG2 cells. Detection of menin and GAPDH is shown on the SDS-PAGE gel. MI-503 was used at 2 mmol/L.






Hep3B cells was markedly reduced upon treatment with MI-503when compared with the DMSO-treated cells (Fig. 2A–D; Sup-plementary Fig. S2A and S2B). Furthermore, we have also eval-uated the effect of MI-503 on sphere formation in HepG2 andHep3B cell lines, both of which were previously shown to retainthe capacity of sphere formation (33, 34), a propensity of cancerstem cells (35). Treatment with MI-503 strongly inhibited sphereformation in both cell lines as indicated by markedly reducedsphere numbers (Fig. 2E and G) and substantially smaller spheresizes when compared with DMSO-treated cells (Fig. 2F and H).Overall, all these data confirm strong effect of theMI-503menin–MLL inhibitor in HCC cell lines, supporting a potential thera-peutic value of menin–MLL inhibitors in HCC.

MI-503 affects expression of genes linked to HCCPrevious studies have suggested that menin promotes liver

tumorigenesis by regulating the expression and function of YAP1,an important "driver gene" in HCC, through an epigenetic mech-anism involving the H3K4me3 histone mark (10). To investigatewhether pharmacologic inhibition of the menin–MLL1 interac-tion affects YAP1 expression, we treated HepG2 cells withMI-503and assessed YAP1 expression by qRT-PCR. We found a dose-dependent reduction in YAP1 expression after 6 days of treatmentwith MI-503, but this effect was relatively modest, with onlyapproximately 40% downregulation of YAP1 observed followingtreatment with 2 mmol/L MI-503 (Fig. 3A). Furthermore, down-regulation of YAP1 expression did not becomemore pronouncedafter prolonged (13 days) treatment of HepG2 cells with MI-503(Fig. 3A).

To further investigate the effect of menin–MLL1 inhibition ongene expression changes in HCC cells, we performed the RNAsequencing (RNA-seq) studies inHepG2 cells following treatmentwith MI-503. We found 291 and 552 genes down- and upregu-lated, respectively, which showed at least 2-fold change in theexpression level following treatment with MI-503 (Supplemen-tary Table S2). We then have performed the GSEA analysis andfound significant enrichment for genes up anddownregulated in asubclass of HCCwith activatedCTNNB1 gene encoding b-catenin(36). A set of genes up- and downregulated upon treatment withMI-503 is, respectively, down and upregulated in this subtype ofHCC (Fig. 3B andC). TheCTNNB1 gene encodes b-catenin and itsmutations lead to the activation of Wnt signaling, which plays anessential role in development of HCC by controlling cell prolif-eration, apoptosis, cell cycle, and motility (37–39). Mutations inCTNNB1 are highly prevalent in HCC, representing the secondmost frequently mutated gene in HCC (40). Indeed, the HepG2cell line represents the liver cancer cell line characterized byactivated Wnt signaling and expresses a truncated b-catenin lack-ing the Ser/Thr domain that regulates its degradation (ref. 41;Supplementary Table S3). The observation that MI-503 reversesgene expression signature in HCCwith activating CTNNB1muta-tions supports the potential application of menin–MLL1 inhibi-tors in HCC with activated Wnt signaling pathway.

The RNA-seq data for HepG2 cells treated withMI-503 showedonly a minor reduction (�20%) in the YAP1 expression, whichdid not reach statistical significance (Supplementary Table S2);this was likely due to a lower sensitivity of this method comparedwith qRT-PCR (see above). Interestingly, within the top genesstrongly downregulated following treatment with MI-503, wefound several genes known to promote proliferation ormigrationof HCC cells, including PEG10, SLC38A4, and SEMA3C (Supple-

mentary Table S2). These results were further validated by theqRT-PCR studies in both HepG2 and Hep3B cells, demonstratinga strong reduction in the expression level of these genes followingtreatment with MI-503 (Fig. 3D and E).

The PEG10 (Paternally Expressed Gene 10) gene is notexpressed in normal adult liver (24) but is highly overexpressedin HCC (24, 25). Indeed, the Oncomine (30) analysis has con-firmed the high overexpression of PEG10 in HCC in different setsof patient samples when compared with normal liver samples(Fig. 3F). Furthermore, significant correlation of PEG10 andMEN1 expression was found in the HCC patient samples but notin normal liver samples (Fig. 3G and H), suggesting a positiveregulation of PEG10 by menin. Importantly, PEG10 promotescancer cell growth in HCC and is associated with poor patientsurvival andwith tumor recurrence (24, 25). Therefore, inhibitionof HCC cell growth induced byMI-503 (Fig. 1C) could, at least inpart, result from the downregulation of PEG10. As mentionedabove, treatment of HCC cells with MI-503 also leads to down-regulation of both SLC38A4, a gene activated in HCC that confersgrowth and survival advantages in the premalignant and malig-nant lesions (42), and SEMA3C (semaphorin 3C), which plays animportant role in migration of HCC cells (ref. 43; Fig. 3D and E;Supplementary Table S2). These results suggest that pharmaco-logic inhibition of the menin–MLL interaction can block hepa-tocellular carcinoma by its effects on pathways associatedwith theproliferation and migration of HCC cells.

Interestingly, among genes markedly upregulated upon treat-ment of HepG2 cells with MI-503 we found GDF15 (GrowthDifferentiation Factor 15; Supplementary Table S2). The qRT-PCRstudies performed in HepG2 and Hep3B cells demonstratedapproximately 3-fold increase in GDF15 expression followingtreatment with MI-503, and this effect was dose dependent (Fig.3D and E). Downregulation ofGDF15was previously found to beassociated with HCC development, while restoration of GDF15level attenuated the progression ofHCC (44); however, the role ofthis gene in HCC seems to be quite complex (45). Nevertheless,we selected this gene as a positive control in the gene expressionstudies as its level increases following menin–MLL1 inhibition,but the functional consequences ofGDF15 changes due toMI-503treatment of HCC cells still need further investigation.

PEG10 is a direct target of menin–MLL1 complexThe finding that treatment ofHCC cells withMI-503 resulted in

downregulation of PEG10 prompted us to perform chromatincoimmunoprecipitation (ChIP) experiments in HepG2 cells todetermine whether the menin–MLL1 complex regulates PEG10expression through a direct binding to this gene. We first assessedmenin binding to PEG10 and found strong enrichment of meninat two sites (P-B and P-C; Fig. 4A and B). We then assessed theeffect of MI-503 on the binding of the menin–MLL1 complex toPEG10 at the sites with highest menin occupancy (Fig. 4A and B).We observed a strong, dose-dependent reduction in both meninand MLL1 binding to the PEG10 promoter after 6 days of treat-ment with MI-503 (Fig. 4C and D). MLL1 is a histone methyl-transferase that catalyzes H3K4 tri-methylation, representing achromatin activation mark. Therefore, we assessed whetherreduced binding of the menin–MLL1 complex to PEG10 inducedby MI-503 affects the H3K4 tri-methylation mark at those sites.We found that MI-503 treatment resulted in marked reduction inH3K4me3 at both sites on PEG10 (Fig. 4C and D) consistent withthe reduced expression of PEG10 (Fig. 3D). These results support

Kempinska et al.





an epigenetic regulation of PEG10 by the menin–MLL1 complexand demonstrate that pharmacologic inhibition of the menin–MLL1 interaction releases this complex from PEG10 and leads to areduction in H3K4 trimethylation and downregulation of PEG10expression.

Sorafenib sensitizes HCC cells to MI-503 treatmentSorafenib, a multikinase inhibitor of tyrosine protein kinases

with a validated therapeutic activity against HCC, has beenapproved for clinical use in patients with unresectable hepatocel-lular carcinoma (1). We explored whether sorafenib would sen-

sitize HCC cells to pharmacologic inhibition of the menin–MLL1interaction by testing a combination of sorafenib and MI-503.Both sorafenib and MI-503 were used at concentrations requiredfor effective growth inhibition in HCC cells (ref. 4; Fig. 5A and C).The inhibitory effect ofMI-503onHepG2 cell growthwas stronglyenhanced by a combination with sorafenib (Fig. 5A). The com-bination indices (CIs) calculated for the simultaneous treatmentofHepG2 cells withMI-503 and sorafenib ranged from 0.45 to 0.66,suggesting strong synergistic effect (Fig. 5A and B). Similarly, thecombination of MI-503 and sorafenib resulted in a more pro-nounced inhibition of cell growth in Hep3B cells when compared

Figure 2.

MI-503 blocks sphere formation andcell migration in HCC cell lines. A–D,Inhibition of cell migration in HepG2(A and B) and Hep3B (C and D) cellsupon treatment with MI-503 measuredby the wound closure assay. Data in Aand C represent mean of duplicates �SD. � , P < 0.05; �� , P < 0.01. E and G,Quantification of spheres in the sphereformation assay performed in HepG2(E) and Hep3B (G) cells upon 14 days oftreatment with MI-503 and DMSO. Datarepresent mean of duplicates � SD. Fand H, Pictures of spheres upontreatment of HepG2 (F) and Hep3B (H)cells with MI-503 or DMSO.






Figure 3.

Menin–MLL inhibitor inhibits genes involved in proliferation andmigration of HCC cells.A,Quantitative RT-PCR analysis of YAP1 expression performed in HepG2 cellsafter 6 and 13 days of treatment with MI-503 or DMSO. Data represent mean of duplicates � SD. B and C, Gene Set Enrichment Analysis (GSEA) of genesdownregulated (B) or upregulated (C) upon treatmentwithMI-503 in HepG2 cells as comparedwith genes from theWnt signaling overexpressed in patient samples.The heatmaps show genes comprising the leading edge of the GSEA plots. Red, high expression; blue, low expression. Triplicate samples were used forglobal gene expression studies.D andE,Quantitative RT-PCR analysis ofPEG10, SEMA3C, SLC38A4, andGDF15 expressionperformed inHepG2 (D) orHep3B (E) cellsafter 6 days of treatment with MI-503 or DMSO. Data represent mean of triplicates � SD. Expression of genes in A, D, and E was normalized to GAPDH andreferenced to DMSO-treated cells. F, Expression level of PEG10 in normal liver samples comparedwith hepatocellular carcinoma as obtained from the analysis of twodatasets (Roessler liver, Roessler liver 2) in the Oncomine database. G and H, Correlation of PEG10 with MEN1 expression in HCC (G) or normal liver samples(H). Gene expression is shown as log2 median-centered intensity. NES, normalized enrichment score; FDR, false discovery rate.

Kempinska et al.





Figure 4.

Epigenetic regulation of PEG10 by menin-MLL1 in HCC. A, Diagram showing the location of primers at the promoter and downstream regions of PEG10. Genomiccoordinates are provided in Supplementary Methods. B, Menin binding to different regions (P-A – P-D) of PEG10 obtained from the ChIP experimentperformed in HepG2 cells. C and D, ChIP experiments performed in HepG2 cells upon 6 days of treatment with various concentrations of MI-503 or DMSO to detectmenin and MLL1 binding as well as H3K4me3 on PEG10. Real-time PCR was performed on the precipitated DNAs with P-B (C) and P-C (D) primers for PEG10genomic regions. IgG was used as a control. Data represent mean of triplicates � SD.






with treatment with either compound individually (Fig. 5C). Thecalculated CI values (<0.66) support synergistic effect of MI-503and sorafenib (Fig. 5C and D). Overall, these results demonstratethat sorafenib sensitizes HCC cells to the pharmacologic inhibi-tion of the menin–MLL1 interaction.

MI-503 inhibits tumor growth in vivo in HCC xenograft modelsThe pronounced effects of MI-503 observed in HCC cell lines

promptedus to assess the therapeutic potential of themenin–MLLinhibitor in in vivomodels of HCC. We have recently shown thatMI-503 has a favorable pharmacokinetic profile in mice and hasdemonstrated strong in vivo efficacy in mice models of MLLleukemia (18) and in castration-resistant prostate cancer (19),which emphasizes its suitability as a candidate for in vivo studies.Here, we used MI-503 both as a single agent and in combinationwith sorafenib to assess the in vivo efficacy of the menin–MLL1

inhibition in two mouse models of HCC: HepG2 and Hep3Bxenograftmodels. TheHepG2orHep3B cells were implanted intothe flank of athymic nude mice. Treatment with MI-503 and/orsorafenib was then initiated when the tumors reached approxi-mately 100 mm3 volume and was continued until the volume ofthe tumor in the vehicle-treated mice reached approximately1,200 mm3 (2–3 weeks). Once-daily intraperitoneal administra-tion of MI-503 at a 35 mg/kg dose caused a strong reduction(>50%) in the tumor growth inbothHepG2andHep3B xenograftmodels (Fig. 6A and B). Furthermore, an 85% inhibition of thetumor growth was observed in both xenograft models when MI-503 was combined with sorafenib (Fig. 6A and B). Importantly,no substantial signs of toxicity (e.g., no significant changes inmouse body weight or liver enzymes level), were observed in themice during the treatment (Supplementary Fig. S3). These resultssupport a potential therapeutic value ofmenin–MLL inhibitors in

Figure 5.

Sorafenib sensitizedHCC cells to the treatmentwithMI-503.A,MTT cell viability assayperformed after 4 days of treatment of HepG2 cellswithMI-503, sorafenib, andboth agents tested simultaneously. Data represent mean of quadruplicates � SD. B, Isobologram obtained from analysis of the combination of MI-503 andsorafenib in HepG2 cells after 4 days of treatment. Combinatorial indices (CI < 1) indicate synergistic effect between these two agents in HepG2 cells. C, MTT cellviability assay performed after 5 days of treatment of Hep3B cells with MI-503, sorafenib, or both agents tested simultaneously. Data represent mean ofquadruplicates � SD. D, Isobologram obtained from analysis of the combination of MI-503 and sorafenib in Hep3B cells after 5 days of treatment. Combinatorialindices (CI < 1) indicate synergistic effect between these two agents in Hep3B cells.

Kempinska et al.





HCC. As sorafenib alone leads to >50% tumor growth inhibitionin both xenograftmodels (Fig. 6A and B) it remains to be exploredwhether lower doses of both compounds could result in evenstronger combinatorial effect in vivo.

We next examined gene expression in the tumor samplescollected from the HepG2 xenograft mice treated with MI-503,sorafenib, combination of both agents or vehicle. The tumorsamples collected frommice treated withMI-503 as a single agent

showed a marked reduction in the expression level of PEG10 andSLC38A4, which are both growth-promoting genes in HCC cells(Fig. 6C). Upregulation of GDF15 was also observed in MI-503–treated mice when compared with vehicle-treated control mice(Fig. 6C). These results were consistent with the on target activityof MI-503 and gene expression studies performed in vitro in theHepG2 and Hep3B cells (Fig. 3D and E). In contrast, we did notobserve statistically significant effect on the expression level of

Figure 6.

In vivo efficacy of MI-503 in HCC models.A and B, In vivo inhibition of the tumorgrowth by MI-503 (35 mg/kg, i.p.),sorafenib (20 mg/kg or 40 mg/kg inHepG2 and Hep3B xenografts,respectively, p.o.), or combination of bothagents relative to vehicle control inHepG2 (A) and Hep3B (B)mice xenograftmodels, n ¼ 8–9 mice per group. Micewere treated once daily at the dosesindicated above. Error bars representSEM. C, Expression of PEG10, SLC38A4andGDF15measured by qRT-PCR of RNAextracted from tumor samples harvestedat the endpoint of treatment with MI-503,sorafenib, combination of both agents orvehicle in HepG2 xenograft model (A).PEG10, SLC38A4, and GDF15 transcriptslevels are normalized to the meantranscript level in tumors from vehicle-treatedmice;n¼ 5 per group.P<0.05 areconsidered significant. NS, not significant.D, Menin interaction with the MLL1histone methyltransferase complex leadsto increase in the H3K4 tri-methyl markon PEG10 and possibly other genesrelevant to HCC resulting in increasedexpression of these genes (left).Inhibition of the menin–MLL1 interactionby MI-503 results in reduced H3K4me3and repression of PEG10 and possiblyother genes relevant to HCC (right).






PEG10, SLC38A4, or GDF15 in the tumor samples isolated frommice treated with sorafenib alone (Fig. 6C). These results indicatea differentmechanism of action for sorafenib andMI-503 inHCCcells. Interestingly, combinatorial treatment of MI-503 and sor-afenib in mice resulted in a similar trend in the gene expressionchanges as observed for MI-503 alone (especially for the down-regulated genes), but the effects are less pronounced and did notreach statistical significance (Fig. 6C). Taken together, the reduc-tion in the tumor growth in the in vivomodels of HCC induced byMI-503 correlated with the decrease in the expression level ofgenes linked to HCC, thereby validating the on target mechanismof action of MI-503. These results demonstrate that pharmaco-logic inhibition of the menin–MLL interaction and/or its com-bination with sorafenib could represent an effective approach forHCC treatment.

DiscussionHCC represents an aggressive and incurable cancer that requires

new therapies for effective patient treatment. Menin is upregu-lated in HCC (Fig. 1A) and a high level of menin expressioncorrelates with poor patient survival, emphasizing an importantrole of menin in HCC (10). As menin interaction with MLL1 wasindicated to play an important role in the development of HCC,we hypothesized that pharmacologic inhibition of this interac-tion could represent a new therapeutic approach inHCC.Here,weestablished that MI-503, a potent small-molecule inhibitor of themenin–MLL1 interaction, has a pronounced antitumor effect inboth in vitro and in vivo models of HCC. Treatment with MI-503results in effective killing of HCC cells and reduces sphere for-mation and cell migration. The combination of MI-503 withsorafenib, multikinase inhibitor currently used in the clinic forHCC treatment, synergistically enhanced the effects of MI-503 onproliferation of HCC cells. Furthermore, in vivo application ofMI-503 resulted inmarked reduction in tumor growth in HepG2 andHep3B xenograft models, and these effects were enhanced bycombination of MI-503 with sorafenib. Overall, our studiesdemonstrate that pharmacologic inhibition of the menin–MLLinteraction has a pronounced effect in HCC models, both in vitroand in vivo, and this approach might be promising as therapy forHCC patients.

Previous studies have suggested that menin function in livertumorigenesis is associated with transcriptional activation ofYAP1, an important oncogene in HCC (10, 15). However, herewe found that pharmacologic inhibition of the menin–MLL1interaction had only a modest effect on the YAP1 expression inHepG2 cells. Instead, we found strong downregulation of othergenes involved in HCC cell growth or migration, includingPEG10, SLC38A4, and SEMA3C, following treatment with theMI-503 menin–MLL inhibitor. We further validated that MI-503reduces the binding of menin and MLL1 to PEG10 and decreasesthe H3K4me3 level at PEG10, providing an important mechanis-tic link between PEG10 and the menin–MLL1 axis in HCC, Fig.6D. Furthermore, global gene expression studies have identifiedthat MI-503 affects expression of genes associated with a subclassof HCC with mutations in CTNNB1, which encodes b-catenin,thereby providing a link to the Wnt signaling pathway that isknown to play a crucial role in a subset of HCC (37–39). Overall,our studies demonstrate that the role of menin in HCC is muchmore complex than the transcriptional regulation of YAP1 andHippo pathway, as suggested previously (10). The menin–MLL1

complex participates in epigenetic activation of several genesinvolved in HCC pathogenesis, rather than regulating a specificpathway in HCC.

We have previously demonstrated strong antitumor effects oftheMI-503menin–MLL1 inhibitor in in vitro and in vivomodels ofMLL leukemia (18), prostate cancer (19), and Ewing sarcoma(46). In addition, our earlier menin–MLL1 inhibitor, MI-2, alsostrongly inhibited tumor cell growth in pediatric gliomas withH3.3K27Mmutations (47) and downregulated genes involved inthe estrogen receptor–positive breast cancer (48). In the studypresented here, we demonstrate that pharmacologic inhibition ofthe menin–MLL1 interaction by MI-503 leads to the effectivekilling of HCC cells and pronounced in vivo tumor growthinhibition, providing an another example of the antitumor effectresulting from blocking the menin–MLL1 interaction. The resultsof these studiesmight suggest a novel andpromising treatment forHCCpatients by using themenin–MLL1 inhibitors either as singleagents or in combination with known drugs, including sorafenib.While menin–MLL1 inhibitors may represent valuable anticanceragents, further studies are needed to assess inhibition of themenin–MLL1 interaction in the context of tumor suppressorfunction of menin in endocrine tissues (49). Although loss ofmenin results in MEN1 tumors, manyMEN1missense mutationsare outside of theMLL-binding site onmenin (50), suggesting thatother menin interactions may be required for the tumor suppres-sor activity of menin. Clinical studies are needed to address theutility of menin–MLL1 inhibitors in HCC and other cancers.

Disclosure of Potential Conflicts of InterestT. Cierpicki reports receiving a commercial research grant, has ownership

interest (including patents), and is a consultant/advisory board member forKuraOncology. J. Grembecka reports receiving a commercial research grant, hasownership interest (including patents), and is a consultant/advisory boardmember forKuraOncology, Inc.Nopotential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: B. Malik, T. Cierpicki, J. GrembeckaDevelopment of methodology: B. Malik, T. CierpickiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Kempinska, B. Malik, S. Klossowski, S. Shukla,H. MiaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Kempinska, B. Malik, J. Wang, J. GrembeckaWriting, review, and/or revision of the manuscript: B. Malik, S. Klossowski,S. Shukla, J. Wang, T. Cierpicki, J. GrembeckaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Malik, D. Borkin, J. WangStudy supervision: T. Cierpicki, J. Grembecka

AcknowledgmentsWe thank Dr. Ilona Kryczek, University of Michigan (Ann Arbor, MI), for

providing MHCC97, PLC/PRF/5, SNU449, and SNU423 human HCC cell linesfor this project and Dr. Elizabeth Lawlor, University of Michigan (Ann Arbor,MI), for providing the ASC52 cell line. The mouse work was performed underoversight of UCUCA at the University of Michigan (Ann Arbor, MI). This workwas funded by the NIH grants R01 (1R01CA160467, to J. Grembecka) and R01(1R01CA200660, to J. Grembecka), LLS Scholar (1215-14, to J. Grembecka),and American Cancer Society grant (RSG-13-130-01-CDD, to J. Grembecka).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 22, 2017; revised September 14, 2017; accepted October 27,2017; published OnlineFirst November 15, 2017.

Kempinska et al.





References1. Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcino-

ma. Hepatology 2008;48:1312–27.2. Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet 2003;

362:1907–17.3. Balogh J, Victor D III, Asham EH, Burroughs SG, Boktour M, Saharia A,

et al. Hepatocellular carcinoma: a review. J Hepatocell Carcinoma 2016;3:41–53.

4. Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, et al. Sorafenibblocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, andinduces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 2006;66:11851–8.

5. Feo F, Pascale RM, Simile MM, De Miglio MR, Muroni MR, Calvisi D.Genetic alterations in liver carcinogenesis: implications for new preventiveand therapeutic strategies. Crit Rev Oncog 2000;11:19–62.

6. Bosch FX, Ribes J, Diaz M, Cleries R. Primary liver cancer: worldwideincidence and trends. Gastroenterology 2004;127:S5–S16.

7. Takenaka K, Kawahara N, Yamamoto K, Kajiyama K, Maeda T, Itasaka H,et al. Results of 280 liver resections for hepatocellular carcinoma. Arch Surg1996;131:71–6.

8. Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, et al. Efficacy and safetyof sorafenib in patients in the Asia-Pacific region with advanced hepato-cellular carcinoma: a phase III randomised, double-blind, placebo-con-trolled trial. Lancet Oncol 2009;10:25–34.

9. Bertot LC, Sato M, Tateishi R, Yoshida H, Koike K. Mortality andcomplication rates of percutaneous ablative techniques for thetreatment of liver tumors: a systematic review. Eur Radiol 2011;21:2584–96.

10. Xu B, Li SH, Zheng R, Gao SB, Ding LH, Yin ZY, et al. Menin promoteshepatocellular carcinogenesis and epigenetically up-regulates Yap1 tran-scription. Proc Natl Acad Sci U S A 2013;110:17480–5.

11. Grembecka J, Belcher AM, Hartley T, Cierpicki T. Molecular basis of themixed lineage leukemia-menin interaction: implications for targetingmixed lineage leukemias. J Biol Chem 2010;285:40690–8.

12. Yokoyama A, Somervaille TC, Smith KS, Rozenblatt-Rosen O, MeyersonM, Cleary ML. The menin tumor suppressor protein is an essentialoncogenic cofactor for MLL-associated leukemogenesis. Cell 2005;123:207–18.

13. Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G.Genome regulation by polycomb and trithorax proteins. Cell 2007;128:735–45.

14. Milne TA, Hughes CM, Lloyd R, Yang Z, Rozenblatt-Rosen O, Dou Y,et al. Menin and MLL cooperatively regulate expression of cyclin-dependent kinase inhibitors. Proc Natl Acad Sci U S A 2005;102:749–54.

15. Zender L, Spector MS, Xue W, Flemming P, Cordon-Cardo C, Silke J, et al.Identification and validation of oncogenes in liver cancer using an inte-grative oncogenomic approach. Cell 2006;125:1253–67.

16. Zhou D, Conrad C, Xia F, Park JS, Payer B, Yin Y, et al. Mst1 and Mst2maintain hepatocyte quiescence and suppress hepatocellular carcinomadevelopment through inactivation of the Yap1 oncogene. Cancer Cell2009;16:425–38.

17. Zhao B, Li L, Lei Q, Guan KL. The Hippo-YAP pathway in organ sizecontrol and tumorigenesis: an updated version. Genes Dev 2010;24:862–74.

18. Borkin D, He S, Miao H, Kempinska K, Pollock J, Chase J, et al. Pharma-cologic inhibition of theMenin-MLL interactionblocks progression ofMLLleukemia in vivo. Cancer Cell 2015;27:589–602.

19. Malik R, Khan AP, Asangani IA, Cieslik M, Prensner JR, Wang X, et al.Targeting theMLL complex in castration-resistant prostate cancer. NatMed2015;21:344–52.

20. Gao SB, Xu B, Ding LH, ZhengQL, Zhang L, ZhengQF, et al. The functionaland mechanistic relatedness of EZH2 and menin in hepatocellular carci-noma. J Hepatol 2014;61:832–9.

21. He S, Malik B, Borkin D, Miao H, Shukla S, Kempinska K, et al. Menin-MLL inhibitors block oncogenic transformation by MLL-fusion pro-teins in a fusion partner-independent manner. Leukemia 2016;30:508–13.

22. Borkin D, Pollock J, Kempinska K, Purohit T, Li X, Wen B, et al. Propertyfocused structure-based optimization of small molecule inhibitors of the

protein-protein interaction between menin and mixed lineage leukemia(MLL). J Med Chem 2016;59:892–913.

23. Grembecka J, He S, Shi A, Purohit T, Muntean AG, Sorenson RJ, et al.Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins inleukemia. Nat Chem Biol 2012;8:277–84.

24. BangH,Ha SY,Hwang SH, ParkCK. Expression of PEG10 is associatedwithpoor survival and tumor recurrence in hepatocellular carcinoma. CancerRes Treat 2015;47:844–52.

25. IpWK, Lai PB,WongNL, Sy SM, Beheshti B, Squire JA, et al. IdentificationofPEG10 as a progression related biomarker for hepatocellular carcinoma.Cancer Lett 2007;250:284–91.

26. Liu L, Ren ZG, Shen Y, Zhu XD, Zhang W, Xiong W, et al. Influence ofhepatic artery occlusion on tumor growth and metastatic potential in ahuman orthotopic hepatoma nude mouse model: relevance of epithelial-mesenchymal transition. Cancer Sci 2010;101:120–8.

27. Chen R, Dong Y, Xie X, Chen J, Gao D, Liu Y, et al. Screeningcandidate metastasis-associated genes in three-dimensional HCCspheroids with different metastasis potential. Int J Clin Exp Pathol2014;7:2527–35.

28. MartinezMolina D, Jafari R, IgnatushchenkoM, Seki T, Larsson EA, Dan C,et al. Monitoring drug target engagement in cells and tissues using thecellular thermal shift assay. Science 2013;341:84–7.

29. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: thecombined effects of multiple drugs or enzyme inhibitors. Adv EnzymeRegul 1984;22:27–55.

30. Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, et al.ONCOMINE: a cancer microarray database and integrated data-miningplatform. Neoplasia 2004;6:1–6.

31. Roessler S, Jia HL, Budhu A, Forgues M, Ye QH, Lee JS, et al. A uniquemetastasis gene signature enables prediction of tumor relapse in early-stagehepatocellular carcinoma patients. Cancer Res 2010;70:10202–12.

32. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient andinexpensive method for analysis of cell migration in vitro. Nat Protoc2007;2:329–33.

33. Cao L, Zhou Y, Zhai B, Liao J, Xu W, Zhang R, et al. Sphere-forming cellsubpopulations with cancer stem cell properties in human hepatoma celllines. BMC Gastroenterol 2011;11:71.

34. Uchida Y, Tanaka S, Aihara A, Adikrisna R, Yoshitake K,Matsumura S, et al.Analogybetween sphere forming ability and stemness of humanhepatomacells. Oncol Rep 2010;24:1147–51.

35. Weiswald LB, Bellet D, Dangles-Marie V. Spherical cancer models in tumorbiology. Neoplasia 2015;17:1–15.

36. Chiang DY, Villanueva A, Hoshida Y, Peix J, Newell P, Minguez B, et al.Focal gains of VEGFA and molecular classification of hepatocellularcarcinoma. Cancer Res 2008;68:6779–88.

37. Laurent-Puig P, Legoix P, Bluteau O, Belghiti J, Franco D, Binot F, et al.Genetic alterations associatedwith hepatocellular carcinomas define distinctpathways of hepatocarcinogenesis. Gastroenterology 2001;120:1763–73.

38. Liu LJ, Xie SX, Chen YT, Xue JL, Zhang CJ, Zhu F. Aberrant regulation ofWntsignaling in hepatocellular carcinoma. World J Gastroenterol 2016;22:7486–99.

39. Waisberg J, Saba GT. Wnt-/-beta-catenin pathway signaling in humanhepatocellular carcinoma. World J Hepatol 2015;7:2631–5.

40. Boyault S, RickmanDS, de Reynies A, BalabaudC, Rebouissou S, Jeannot E,et al. Transcriptome classification of HCC is related to gene alterations andto new therapeutic targets. Hepatology 2007;45:42–52.

41. Lachenmayer A, Alsinet C, Savic R, Cabellos L, Toffanin S, Hoshida Y, et al.Wnt-pathway activation in two molecular classes of hepatocellular carci-noma and experimental modulation by sorafenib. Clin Cancer Res2012;18:4997–5007.

42. Kondoh N, Imazeki N, Arai M, Hada A, Hatsuse K, Matsuo H, et al.Activationof a systemAamino acid transporter, ATA1/SLC38A1, in humanhepatocellular carcinoma and preneoplastic liver tissues. Int J Oncol 2007;31:81–7.

43. Liao YL, Sun YM, Chau GY, Chau YP, Lai TC, Wang JL, et al. Identi-fication of SOX4 target genes using phylogenetic footprinting-basedprediction from expression microarrays suggests that overexpression ofSOX4 potentiates metastasis in hepatocellular carcinoma. Oncogene2008;27:5578–89.






44. Yu J, Shen B, Chu ES, Teoh N, Cheung KF, Wu CW, et al.Inhibitory role of peroxisome proliferator-activated receptor gam-ma in hepatocarcinogenesis in mice and in vitro. Hepatology 2010;51:2008–19.

45. Zimmers TA, Jin X, Gutierrez JC, Acosta C, McKillop IH, Pierce RH, et al.Effect of in vivo loss of GDF-15 on hepatocellular carcinogenesis. J CancerRes Clin Oncol 2008;134:753–9.

46. Svoboda LK, Bailey N, Van Noord RA, Krook MA, Harris A, CramerC, et al. Tumorigenicity of Ewing sarcoma is critically dependenton the trithorax proteins MLL1 and menin. Oncotarget 2017;8:458–71.

47. Funato K, Major T, Lewis PW, Allis CD, Tabar V. Use of human embryonicstem cells to model pediatric gliomas with H3.3K27M histone mutation.Science 2014;346:1529–33.

48. Dreijerink KM, Groner AC, Vos ES, Font-Tello A, Gu L, Chi D, et al.Enhancer-mediated oncogenic function of the menin tumor suppressorin breast cancer. Cell Rep 2017;18:2359–72.

49. Chandrasekharappa SC, Teh BT. Functional studies of the MEN1 gene.J Intern Med 2003;253:606–15.

50. MuraiMJ, ChruszczM, ReddyG,Grembecka J, Cierpicki T. Crystal structureof menin reveals binding site for mixed lineage leukemia (MLL) protein.J Biol Chem 2011;286:31742–8.


Kempinska et al.




2018;17:26-38. Published OnlineFirst November 15, 2017.Mol Cancer Ther Katarzyna Kempinska, Bhavna Malik, Dmitry Borkin, et al. Carcinoma

and Blocks HepatocellularPEG10Transcriptional Repression of MLL Interaction Leads to−Pharmacologic Inhibition of the Menin

Updated version

10.1158/1535-7163.MCT-17-0580doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2017/11/15/1535-7163.MCT-17-0580.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/17/1/26.full#ref-list-1

This article cites 50 articles, 11 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/17/1/26To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-17-0580

http://mct.aacrjournals.org/content/suppl/2017/11/15/1535-7163.MCT-17-0580.DC1

http://mct.aacrjournals.org/content/17/1/26.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/17/1/26


Documents

Pharmacologic Inhibition of the Menin MLL Interaction ... · Chromatin immunoprecipitation A total of 1.5 106 HepG2 cells plated in 10-cm dish were treated with DMSO (0.25%) or MI-503